Decoding Transcription Regulatory Mechanisms Associated with Phase Transition Using Total RNA.

New or emerging infectious diseases are commonly caused by pathogens that cannot be readily manipulated or studied under common laboratory conditions. These limitations hinder standard experimental approaches and our abilities to define the fundamental molecular mechanisms underlying pathogenesis. The advance of capped small RNA sequencing (csRNA-seq) now enables genome-wide mapping of actively initiated transcripts from genes and other regulatory transcribed start regions (TSRs) such as enhancers at a precise moment from total RNA.

Transcriptional Analysis of Mycelia and Spherules by RNA Sequencing.

and   are dimorphic fungi that transform from mycelia with internal arthroconidia in the soil to a tissue form known as a spherule in mammals. This process can be recapitulated in vitro by increasing the temperature, CO and changing other culture conditions. In this study, we have analyzed changes in gene expression in mycelia and young and mature spherules.

Interleukin-8 Receptor 2 (IL-8R2)-Deficient Mice Are More Resistant to Pulmonary Coccidioidomycosis than Control Mice.

The pathology of human coccidioidomycosis is granulomatous inflammation with many neutrophils surrounding ruptured spherules, but the chemotactic pathways that draw neutrophils into the infected tissues are not known. We previously showed that formalin-killed spherules (FKS) stimulate mouse macrophages to secret macrophage inflammatory protein 2 (MIP-2), which suggested that CXC ELR+ chemokines might be involved in neutrophil recruitment To test that hypothesis, we intranasally infected interleukin-8R2 (IL-8R2) ()-deficient mice on a BALB/c background with RS. IL-8R2-deficient mice had fewer neutrophils in infected lungs than controls, but unexpectedly the IL-8R2-deficient mice had fewer organisms in their lungs than the control mice.

APX001 and Other Gwt1 Inhibitor Prodrugs Are Effective in Experimental Pneumonia.

Coccidioidomycosis is a systemic fungal infection caused by the inhalation of the arthroconidia of either of two closely related dimorphic fungi, and , that are endemic in the southwestern United States and other areas in the Western Hemisphere. Chronic cavitary pulmonary infections and extrapulmonary sites of infection are very difficult to treat and often require lifelong azole therapy. APX001A is the first in a new class of broad-spectrum antifungal agents that inhibit Gwt1, an enzyme which is required for cell wall localization of glycosylphosphatidylinositol (GPI)-anchored mannoproteins in fungi.

Myeloid Differentiation Factor 88 and Interleukin-1R1 Signaling Contribute to Resistance to Coccidioides immitis.

Rodents are a natural host for the dimorphic pathogenic fungi and , and mice are a good model for human infection. Humans and rodents both express Dectin-1 and Toll-like receptor 2 (TLR2) on myeloid cells, and those receptors collaborate to maximize the cytokine/chemokine responses to spherules (the tissue form of the fungi) and to formalin-killed spherules (FKS). We showed that Dectin-1 is necessary for resistance to pulmonary coccidioidomycosis, but the importance of TLR2 is uncertain.

MD-2 binds cholesterol.

Cholesterol is a structural component of cellular membranes, which is transported from liver to peripheral cells in the form of cholesterol esters (CE), residing in the hydrophobic core of low-density lipoprotein. Oxidized CE (OxCE) is often found in plasma and in atherosclerotic lesions of subjects with cardiovascular disease. Our earlier studies have demonstrated that OxCE activates inflammatory responses in macrophages via toll-like receptor-4 (TLR4).

Neither dectin-2 nor the mannose receptor is required for resistance to Coccidioides immitis in mice.

We investigated the roles of the mannose receptor (MR) and Dectin-2 in resistance to pulmonary coccidioidomycosis in C57BL/6 (B6) mice and in the interaction of myeloid cells with spherules, using B6 mice with targeted mutations in Mrc1 and Clec4n. Spherules are the tissue form of Coccidioides, and we determined that the MR on bone marrow-derived dendritic cells (BMDC) was important for recognition of spherules (formalin-killed spherules [FKS]) and for secretion of interleukin 10 (IL-10) and proinflammatory cytokines in response to FKS by both elicited macrophages and BMDC. Infected MR knockout (KO) mice produced more IL-10 in their lungs than did B6 mice, and MR KO mice also made more protective Th-17 cytokines.

Gene expression in human fungal pathogen Coccidioides immitis changes as arthroconidia differentiate into spherules and mature.

Background: Coccidioides immitis is a dimorphic fungus that causes disease in mammals, including human beings. It grows as a mycelium containing arthroconidia in the soil and in the host arthroconidia differentiates into a unique structure called a spherule. We used a custom open reading frame oligonucleotide microarray to compare the transcriptome of C.

Dectin-1 is required for resistance to coccidioidomycosis in mice.

We assessed the role of Dectin-1 in the immune response to the pathogenic fungus Coccidioides, both in vitro and in vivo, using mice with a targeted mutation in Clec7a. Elicited peritoneal macrophages responded to formalin-killed spherules (FKS) and alkali-treated FKS by secreting proinflammatory cytokines in a Dectin-1- and β-glucan-dependent manner. The responses of bone marrow-derived dendritic cells (BMDC) to the same stimulants were more complex; interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α) secretion was independent of Dectin-1, while IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were largely but not entirely dependent on Dectin-1.

Factors regulated by interferon gamma and hypoxia-inducible factor 1A contribute to responses that protect mice from Coccidioides immitis infection.

Background: Coccidioidomycosis results from airborne infections caused by either Coccidioides immitis or C. posadasii. Both are pathogenic fungi that live in desert soil in the New World and can infect normal hosts, but most infections are self-limited.

The role of reactive oxygen intermediates in experimental coccidioidomycois in mice.

Background: Coccidioidomycosis is usually a self-limited infection in immunocompentent people. In immunocompentent human beings second infections due to Coccidioides are very rare, indicating that recovery from infection results in protective immunity. In experimental animals, immunization with several different proteins or attenuated mutants protects against a virulent challenge.

Susceptibility to Coccidioides species in C57BL/6 mice is associated with expression of a truncated splice variant of Dectin-1 (Clec7a).

Coccidioides posadasii spherules stimulate macrophages to make cytokines via TLR-2 and Dectin-1. We used formalin-killed spherules and 1,3-beta-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and resistant (DBA/2) mouse strains. DBA/2 macrophages produced more TNF-alpha and IL-6 than macrophages from C57BL/6 mice, and the amount of TNF-alpha made was dependent on both TLR2 and Dectin-1.

Characterization of monoclonal antibodies to human soluble MD-2 protein.

Toll-like receptors (TLRs) are mammalian innate immune recognition receptors that are activated by pathogen associated molecular patterns (PAMPs). TLR4 is the signaling molecule of the lipopolysaccharide (LPS) receptor complex. TLR4 associates with its adapter molecule, MD-2, which is absolutely required for LPS-induced activation of TLR4.

Engagement of CD14 mediates the inflammatory potential of monosodium urate crystals.

Phagocyte ingestion of monosodium urate (MSU) crystals can induce proinflammatory responses and trigger acute gouty inflammation. Alternatively, the uptake of MSU crystals by mature macrophages can be noninflammatory and promote resolution of gouty inflammation. Macrophage activation by extracellular MSU crystals involves apparent recognition and ingestion mediated by TLR2 and TLR4, with subsequent intracellular recognition linked to caspase-1 activation and IL-1beta processing driven by the NACHT-LRR-PYD-containing protein-3 inflammasome.

Mutational analysis of membrane and soluble forms of human MD-2.

Toll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria. MD-2 is a 20-25-kDa extracellular glycoprotein that binds to Tolllike receptor 4 (TLR4) and LPS and is a critical part of the LPS receptor. Here we have shown that the level of MD-2 expression regulates TLR4 activation by LPS.

Toll-like receptor 4 protects against lethal Leptospira interrogans serovar icterohaemorrhagiae infection and contributes to in vivo control of leptospiral burden.

The roles of innate immune responses in protection from or pathogenesis of severe leptospirosis remain unclear. We examined the role of Toll-like receptors (TLRs) in mouse infection and macrophage responses to Leptospira. C3H/HeJ mice (TLR4 deficient) and C3H/HeJ-SCID mice, but not C3H/OuJ mice (TLR4 intact), died after intraperitoneal infection with Leptospira interrogans serovar Icterohaemorrhagiae.

Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells.

Lipopolysaccharide (LPS) induces inflammatory activation through TLR4 (toll-like receptor-4)/MD-2 (myeloid differentiation-2)/CD14 (cluster of differentiation-14) complex. Although optimal LPS signaling is required to activate our innate immune systems against gram-negative bacterium, excessive amount of LPS signaling develops a detrimental inflammatory response in gram-negative bacterial infections. Downregulation of surface TLR4 expression is one of the critical mechanisms that can restrict LPS signaling.

Innate immunity to the pathogenic fungus Coccidioides posadasii is dependent on Toll-like receptor 2 and Dectin-1.

Coccidioides posadasii is a pathogenic fungus that causes endemic and epidemic coccidioidomycosis in the deserts of North, Central, and South America. How the innate immune system responds to the organism is not well understood. Here we show that elicited mouse peritoneal macrophages respond to spherules (the tissue form of the fungus) by producing proinflammatory cytokines as measured by quantitative PCR of cellular transcripts and by enzyme-linked immunosorbent assay (ELISA) assays for secreted protein.

Toll-like receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized low-density lipoprotein in macrophages.

Objective: Innate immune responses to oxidized low-density lipoprotein LDL (LDL) regulate the development of atherosclerosis. We demonstrated previously that an early form of oxidized LDL, minimally modified LDL (mmLDL), triggers cytoskeletal rearrangements in macrophages via CD14 and Toll-like receptor 4 (TLR4)/MD-2. Because lipopolysaccharide (LPS) activation of TLR4 leads to proinflammatory gene expression, in this study, we asked whether mmLDL also induced proinflammatory signaling.

Lipopolysaccharide (LPS)-binding protein inhibits responses to cell-bound LPS.

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute phase reactant that may play a dual role in vivo, both potentiating and decreasing cell responses to bacterial LPS. Whereas low concentrations of LBP potentiate cell stimulation by transferring LPS to CD14, high LBP concentrations inhibit cell responses to LPS. One inhibitory mechanism involves the ability of LBP to neutralize LPS by transferring it to plasma lipoproteins, whereas other inhibitory mechanisms, such as the one described here, do not require exogenous lipoproteins.

Minimally modified LDL binds to CD14, induces macrophage spreading via TLR4/MD-2, and inhibits phagocytosis of apoptotic cells.

Minimally modified low density lipoprotein (mmLDL) is a pro-inflammatory and pro-atherogenic lipoprotein that, unlike profoundly oxidized LDL (OxLDL), is not recognized by scavenger receptors and thus does not have enhanced uptake by macrophages. However, here we demonstrate that mmLDL (as well as OxLDL) induces actin polymerization and spreading of macrophages, which results in such pro-atherogenic consequences as inhibition of phagocytosis of apoptotic cells but enhancement of OxLDL uptake. We also demonstrate for the first time that the lipopolysaccharide receptor, CD14, and toll-like receptor-4/MD-2 are involved in these mmLDL effects.

MD-2 binds to bacterial lipopolysaccharide.

Many LPS binding proteins have been described, but the exact nature of the LPS receptors that signal cells remains unclear. MD-2 is a molecule that is found in association with Toll-like receptor 4, which has been shown to be a receptor for LPS. We have produced human MD-2 in baculovirus and tested it for LPS binding.

MD-2 binds to bacterial lipopolysaccharide.

The exact roles and abilities of the individual components of the lipopolysaccharide (LPS) receptor complex of proteins remain unclear. MD-2 is a molecule found in association with toll-like receptor 4. We produced recombinant human MD-2 to explore its LPS binding ability and role in the LPS receptor complex.

Plasma CD14 decreases monocyte responses to LPS by transferring cell-bound LPS to plasma lipoproteins.

CD14, a myeloid cell-surface receptor and soluble plasma protein, binds LPS and other microbial molecules and initiates the innate immune response to bacterial invasion. The blood concentration of soluble CD14 (sCD14) increases during the systemic response to infection. Although high sCD14 blood levels have correlated with increased risk of dying from severe sepsis, sCD14 can diminish cell responses to LPS.

Soluble CD14 enhances membrane CD14-mediated responses to peptidoglycan: structural requirements differ from those for responses to lipopolysaccharide.

The purpose of this study was to identify the functional significance of the binding of soluble CD14 (sCD14) to bacterial peptidoglycan (PGN) and to compare the structural requirements of sCD14 for the binding to PGN and lipopolysaccharide (LPS) and for sCD14-mediated enhancement of PGN- and LPS-induced cell responses. sCD14 did not facilitate the responses of membrane CD14 (mCD14)-negative pre-B 70Z/3 cells to PGN, although it facilitated the responses of these cells to LPS and although mCD14 facilitated the responses of 70Z/3 cells to PGN. sCD14 enhanced mCD14-mediated cell activation by both PGN and LPS, but only the responses to LPS, and not to PGN, were enhanced by LPS-binding protein.